| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2004: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
1.A female attracting pheromone of the red-bellied newt Cynops pyrrhogaster "sodefrin", was considered to act through the olfactory organ of female since attraction to this substance was abolished by bilateral nostril plugging or nerve transection between the nasal cavity and the olfactory bulb. Electrophysiological studies revealed that sodefrin evoked a marked electro-olfactogram (EOG) response when applied to the central region of the vomeronasal epithelium of the female. In order to determine the signal-transduction pathways for sodefrin, measurement of the intracellular concentration of Ca^2 ([Ca^<2+>]i) vomeronasal cells subjected to sodefrin by means of microfluorometry of fura-2 was carried out. Increase in the [Ca^<2+>]i was detected in small number of vomeronasal cells from sexually developed female newts after application of sodefrin.
2.Sodefrin was revealed to be a decapeptide with amino acid sequence SIPSKDALLK. Sequence analysis of the PCR products derived from total RNAs
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from the abdominal gland of individual C. prrhogaster collected from the three different regions of Japan, (Chiba, Niigata and Nara) revealed that the expression of genes encoding sodefrin and its variants varied according to the individuals and the regions. However, genes encoding sodefrin were found to be expressed in all the specimens examined. Interestingly, genes encoding a sodefrin variant, [Val8] sodefrin (SIPSKDAVLK) was expressed exclusively in the newts captured in Nara. Furthermore, the regionally specific pheromone variant [Val^8] sodefrin was isolated from the abdominal glands of male C.prrhogaster captured in Nara, by gel-filtration chromatography and HPLC. Synthetic [Val^8] sodefrin and sodefrin equipotent in terms of attracting females, but [Val^8] sodefrin is unique in that it attracts only females captured in nara area, whereas sodefrin is invariably effective in attracting females from three regions tested. These results suggest the occurrence of geographic variation in the pheromone molecules and the responsiveness to the pheromone.
3.Very recently, by way of gas chromatography-mass spectrometry, we demonstrated the presence of steroidal substances such as pregnenolone, progesterone, androstenedione, testosterone and estradiol in the abdominal gland of the male newt. Furthermore, we confirmed that a considerable amount of pregnenolone and androstenedione, as well as sodefrin, are released from the abdominal gland slices to the medium when incubated for 1hr. As a step to elucidate the role of these released steroidal substances, they were tested for their female-attracting activity employing the method developed by Toyoda et al. (1994). As a result, both pregnenolone and androstenedione revealed to possess a female-attracting activity. The minimum effective amount being 0.4 ng that is contained in 0.02 part of the abdominal gland in both cases. Moreover, mixture of either of these two steroids and sodefrin, amount of which exerted more potent attracting activity than the steroidal fraction or sodefrin alone. The results strongly suggest that both the steroids and the peptide pheromone act together to attract sexually developed females during courtship. Less
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